Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes, in metazoans this central genetic element is determined epigenetically rather than by DNA sequence. With support from this grant and using gene replacement in human cells, we have established that chromatin containing the centromere-specific histone H3 variant CENP-A is the epigenetic mark which acts through a two step mechanism to identify, maintain and propagate human or yeast centromere function indefinitely. In the first step, centromere position is replicated and maintained in a cell cycle-dependent manner by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into histone H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is directed by either CENP-A's amino- or carboxy-terminal tails which serve to recruit inner kinetochore proteins. We will now test and extend the two-step model of epigenetic centromere specification by exploiting the rapid, inducible, degron-mediated protein degradation approach whose utility we established in mammalian cells in the prior grant period. With this, we will determine the importance of CENP-A for maintenance of centromeric chromatin and determine the influence of CENP-B and/or chromatin modifications for 'de novo' CENP-A reloading onto centromeric chromatin. The mechanism(s) will be identified through which the CENP-A amino-terminal tail stabilizes CENP-B binding and nucleates kinetochore assembly. Further, the structure of CENP-A chromatin will be determined across the cell cycle using volume and size measurements with solid-state nanopores. Histone composition of CENP-A-containing chromatin, the lengths and DNA sequences bound by CENP-A or the CENP-T/W/S/X nucleosome-like complex at each cell cycle point will be determined by high-throughput DNA sequencing with and without TALEN-mediated inactivation of both CENP- B alleles. Finally, following our discovery that CENP-A and other components known for essential roles in centromere assembly are rapidly recruited to sites of DNA damage, we will assess the roles of CENP-A and its partners in DNA repair of DNA double strand breaks produced by an inducible/inactivatable, site specific CRISPR/Cas9 nuclease that we have generated. This will include determination of the mechanism of transient CENP-A recruitment to sites of DNA damage, the extent of chromatin remodeling following DNA damage, and dependence of overall repair on CENP-A.